Paclitaxel, an antimitotic agent that promotes microtubule assembly, is one of the most potent anti-cancer drugs in clinical use. Its ability to cure disease, however, is limited by the problem of drug resistance;i.e., many patients who initially respond to paclitaxel suffer relapses and become refractory to further treatment. The goal of this project is characterization of the cellular mechanisms responsible for this phenomenon. Mammalian cell tubulin is encoded by a multigene family that produces distinct gene products, or isotypes. One prominent mechanism of resistance involves mutations in tubulin genes that alter microtubule assembly in a way that counteracts the action of the drug. It is not yet clear, however, which mutations and which tubulin genes are involved. To obtain this information, mammalian cell mutants will be selected for resistance to paclitaxel following transfection of site-directed or randomly mutagenized tubulin cDNAs. Transfected DMA from cells that survive in paclitaxel will be sequenced to identify tubulin mutations responsible for resistance, and changes in microtubule assembly in these cell lines will be examined to identify the mechanism responsible for resistance. Additonal studies will use overexpression or silencing of various tubulin genes to test the hypothesis that altered synthesis of specific tubulin isotypes may also contribute to the drug resistance phenotype. Finally, the microtubule interacting proteins stathmin and MCAK will be overexpressed, silenced, and mutagenized to determine whether changes in these microtubule regulators are capable of conferring resistance to antimitotic drugs. The activity of stathmin and MCAK in cells with varying tubulin composition and tubulin mutations will also be measured to explore the possibility that drug resistance may sometimes occur because of altered tubulin-stathmin or tubulin-MCAK interactions. The nformation obtained from the studies in this proposal will be important for characterizing the mechanism of action of antimitotic drugs, developing assays to detect drug resistant cells in human tumors, and formulating strategies to circumvent drug resistance when it occurs.